Retinoic acid receptor composition

ABSTRACT

A novel retinoic acid receptor is disclosed. The novel receptor is encoded for by cDNA carried on plasmid phRAR1, which has been deposited with the American Type Culture Collection for patent purposes. Chimeric receptor proteins are also disclosed. The chimera are constructed by exchanging functional domains between the glucocorticoid, the mineralocorticoid, the estrogen-related, the thyroid and the retinoic acid receptors. In addition, a novel method for identifying functional ligands for receptor proteins is disclosed. The method, which takes advantage of the modular structure of the hormone receptors and the idea that the functional domains may be interchangeable, replaces the DNA-binding domain of a putative novel receptor with the DNA-binding domain of a known receptor such as the glucocorticoid receptor. The resulting chimeric construction, when expressed in cells, produces a hybrid receptor whose activation of a ligand-(e.g., glucocorticoid) inducible promoter is dependent on the presence of the new ligand. The novel method is illustrated in part by showing that the ligand for the new receptor protein is the retinoid, retinoic acid.

ACKNOWLEDGMENT

This invention was made with government support under a grant from theNational Institutes of Health (Grant No. GM 26444).

RELATED APPLICATION

This is a division of application Ser. No. 276,536, now U.S. Pat. No.4,981,784 filed Nov. 30, 1988, which in turn was a continuation-in-partof U.S. Ser. No. 128,331 filed Dec. 2, 1987, now abandoned.

FIELD OF THE INVENTION

The present invention relates generally to ligand-responsive regulatoryproteins and genes encoding them. More particularly, the presentinvention relates to retinoid related regulatory proteins and genesencoding them, modification of these and other regulatory proteins andgenes by recombinant DNA and other genetic engineering techniques, plususes of the retinoid related regulatory proteins and genes, bothunmodified and modified.

In addition the invention relates to a novel method for identifyingfunctional ligands for ligand-responsive proteins. This method isespecially useful for identifying functional ligand(s) for newlydiscovered receptor proteins. The method is exemplified in part byshowing that a vitamin A related morphogen, retinoic acid, is afunctional ligand for a newly discovered retinoid receptor protein.

BACKGROUND OF THE INVENTION

A central problem in eukaryotic molecular biology continues to beelucidation of molecules and mechanisms that mediate specific generegulation in response to exogenous inducers such as hormones or growthfactors. Although much remains to be learned about the specifics of suchmechanisms, it is known that exogenous inducers such as hormonesmodulate gene transcription by acting in concert with intracellularcomponents, including intracellular receptors and discrete DNA known ashormone response elements or HRE's.

More specifically, it is known that hormones like the glucocorticoid andthyroid hormones enter cells by facilitated diffusion. It is also knownthat the hormones then bind to specific receptor proteins, therebycreating a hormone/receptor complex. The binding of hormone to thereceptor is believed to initiate an alosteric alteration of the receptorprotein. As a result of this alteration, it is believed that thehormone/receptor complex is capable of binding with high affinity tocertain specific sites on the chromatin DNA. Such sites, which arereferred to in the art by a variety of names, including hormone responseelements or HRE's, modulate expression (transcription of RNA) of nearbytarget gene promoters.

A major obstacle to further understanding the specifics of generegulation by exogenous inducers such as hormones has been the lack ofavailability of receptor proteins in sufficient quantity and purity toallow such proteins to be adequately analyzed and characterized. Thissame lack of availability has thwarted the use of receptors indiagnostic assays to determine the presence of exogenous inducers (e.g.,the hormones) in various body fluids and tissues, as well as their useas "prototypes" for engineering chimeric receptor protein analogs.

In an effort to overcome this lack of availability of receptor proteins,U.S. Pat. No. 5,071,773 (U.S. Ser. No. 108,471), which has been assignedto the Salk Institute for Biological Studies, assignee of the presentapplication, discloses cloned genes for a variety of receptor proteins,including glucocorticoid-, thyroid-, mineralocorticoid- and newsteroid-related receptors. U.S. Pat. No. 5,071,773 (U.S. Ser. No.108,471) further discloses detailed biochemical characterization ofthese molecules which shows that the receptor proteins contain discreteDNA- and ligand-binding domains. (Portions of U.S. Ser. No. 108,471 havebeen published; for portions relating to cloning of the glucocorticoidreceptor and characterization of this molecule into discrete domains,see Hollenberg, et al. (1985) and Giguere, et al., (1986); for otherrelated work regarding receptors, see Hollenberg, et al., (1987), Green,et al., (1986), Green and Chambon, (1987), Kumar, et al., (1987),Miesfeld, et al., (1987) and Evans (1988)).

Further with regard to biochemical characterization of the receptors,sequence analysis of the human glucocorticoid receptor gene revealedhomology with the product of the v-erb-A oncogene of avianerythroblastosis virus (AEV) (see Weinberger, et al., (1985)). Thisgroup and others subsequently demonstrated the cellular homolog ofv-erb-A to be the beta thyroid hormone receptor (see Weinberger et al.,(1986) and Sap, et al., (1986)).

The discovery that the DNA-binding domain of the steroid and thyroidhormone receptors is highly conserved raised the question of whetherthis segment might be diagnostic for related ligand inducibletranscription factors. It also raised the question of whether the DNAsequences encoding these domains might be used as hybridization probesto scan the genome for related, but novel, ligand-responsive receptors.Utilizing this approach, our group at the Salk Institute have identifiedseveral new gene products. As is shown in U.S. Ser. No. 108,471, one isthe human aldosterone receptor (hMR, ATCC No. 67201) (see Arriza, etal., (1987) for the published version of this portion of U.S. Pat. No.5,071,773 (U.S. Ser. No. 108,471); a second is a novel thyroid hormonereceptor expressed at high levels in the rat central nervous system (rTRalpha, ATCC No. 67281) (see Thompson, et al., (1987) for the publishedversion of this portion of U.S. Pat. No. 5,071,773 (U.S. Ser. No.108,471).

This disclosure describes the isolation and characterization of a clonedfull-length cDNA encoding a novel retinoid receptor protein withhomology to the DNA-binding and ligand-binding domains of the steroidand thyroid hormone receptors. In addition the construction andcharacterization of chimeric receptors made by "swapping" functionaldomains between the glucocorticoid, the mineralocorticoid, the thyroid,the estrogen-related, and the retinoic acid receptors is described.These chimeric receptors have hybrid functional characteristics based onthe "origin" of the "parental" DNA-binding and ligand-binding domainsincorporated within the chimeras. For example, if the DNA-binding domainin the chimeric receptor is a retinoic acid receptor DNA-binding domain(i.e., is obtained from wild-type retinoic acid receptor or is a mutantthat contains the functional elements of retinoic acid DNA-bindingdomain), then the chimera will have DNA-binding propertiescharacteristic of a retinoic acid receptor. The same is true of theligand-binding domain. If the ligand-binding domain in the chimericreceptor binds to thyroid hormone, then the chimera will haveligand-binding properties characteristic of a thyroid hormone receptor.

This disclosure also describes a new method for identifying functionalligands for ligand-responsive receptor proteins. The method isillustrated by showing (1) that the retinoid, retinoic acid and itsmetabolic precurser, retinol, are functional ligands for the newlydiscovered receptor protein, and (2) that the DNA- and ligand-bindingdomains determine the functional characteristics of the chimericreceptors.

BRIEF DESCRIPTION OF THE DRAWINGS

The following is a brief description of the drawings. More detaileddescriptions are found in the section of the specification labeled,"Detailed Description of the Drawings".

FIG. 1 (A and B) is a drawing which shows the DNA nucleotide sequenceand the primary protein sequence of phRARα. FIG. 1A shows the compositestructure of phRARα aligned with a line diagram of some restrictionendonuclease cleavage sites. FIGS. 1B-1, 1B-2 and 1B-3 show the completenucleotide sequence of phRARα and its primary amino acid sequence.

FIG. 2 (A and B) is composed of a drawing and a blot. FIG. 2A is adrawing which illustrates construction of the chimeric receptor hRGR.FIG. 2B is a blot which illustrates induction of CAT activity byretinoic acid.

FIG. 3 (A and B) is composed of two graphs. FIG. 3A is a graphillustrating dose-response to retinoids. FIG. 3B is a bar graphillustrating retinoic acid binding to cytosol extracts of transfectedCOS-1 cells.

FIG. 4 (A and B) shows a Southern blot analysis of human genomic DNA.FIG. 4A shows digested human placenta DNA hybridized under stringentconditions; FIG. 4B shows the same DNA hybridized under non-stringentconditions.

FIG. 5 shows a Northern blot analysis of retinoic acid receptor mRNA inrat and human tissues.

FIG. 6 is a schematic drawing which shows a comparison of hGR, hRR andhT₃ Rβ.

FIG. 7 is a schematic diagram of a generalized steroidthyroidretinoicacid receptor gene.

FIG. 8 (1 and 2) is a schematic drawing that shows amino acid comparisonof members of the steroid hormone receptor superfamily.

FIG. 9 is a schematic drawing that shows the structure and activity ofchimeric thyroid/glucocorticoid receptors.

DEFINITIONS

In the present specification and claims, reference will be made tophrases and terms of art which are expressly defined for use herein asfollows:

As used herein, the generic term "retinoids" means a group of compoundswhich includes retinoic acid, vitamin A (retinol) and a series ofnatural and synthetic derivatives that can exert profound effects ondevelopment and differentiation in a wide variety of systems.

As used herein, species are identified as follows: h, human; r, rat; m,mouse; c, chicken; and d, Drosophilia.

As used herein, "steroid hormone superfamily of receptors" refers to theclass of related receptors comprised of glucocorticoid,mineralocorticoid, progesterone, estrogen, estrogen-related, vitamin D₃,thyroid, v-erb-A, retinoic acid and E75 (Drosophilia) receptors. SeeEvans (1988) and the references cited therein.

As used herein, RR and RAR both mean retinoic acid receptor. Theacronyms, hRR and hRAR, mean human retinoic acid receptor. The DNAreferred to as phRARα codes for human retinoic acid receptor alpha.hRARα is encoded by deposited phRARl which has been accorded ATCC No.40392. The DNA referred to as hRARβ encodes human retinoic acid receptorbeta. See Brand et al., (1988).

As used herein, GR means glucocorticoid receptor. The DNA referred to ashGR codes for human glucocorticoid receptor GR. hGR is encoded bydeposited pRShGR which has been accorded ATCC No. 67200.

As used herein, MR means mineralocorticoid receptor. The DNA referred toas hMR codes for human mineralocorticoid receptor MR. hMR is encoded bydeposited pRShMR which has been accorded ATCC No. 67201.

As used herein, TR means thyroid receptor. TRalpha and TRbeta refer tothe alpha and beta forms of the thyroid receptor. The DNA's referred toas c-erb-A, herb-A 8.7, peA101, rbeA12, and hFA8 all code for thyroidreceptors. Plasmid pherb-A 8.7 encodes hTRα; it has been deposited forpatent purposes and accorded ATCC No. 40374. Plasmid peA101 encodeshTRβ; it has been deposited for patent purposes and accorded ATCC No.67244. Plasmid rbeA12 encodes rTRα; it has been deposited for patentpurposes and accorded ATCC No. 67281. Plasmid phFA8 encodes a partialclone of hTRα that has a deletion in the "ligand-binding" region of theclone (i.e., the DNA that codes for the carboxy terminal end of thereceptor protein). Plasmid phFA8 has been accorded ATCC No. 40372.

As used herein, ERR means estrogen-related receptor. The acronyms, hERR1and hERR2 refer to human estrogen-related receptors 1 and 2. Thesereceptors are more related to steroid receptors than to the thyroidreceptors, yet they do not bind any of the major classes of knownsteroid hormones (Giguere, et al, 1988). hERR1 is encoded by depositedplasmids pE4 and pHKA, which have been accorded ATCC Nos. 67309 and67310, respectively. (Neither pE4 or pHKA are complete clones; hERR1 isconstructed by joining segments from both clones.) hERR2 is encoded bydeposited plasmid phH3 which has been accorded ATCC No. 40373.

As used herein, VDR means vitamin D₃ receptor.

As used herein, MTV means mammary tumor virus; MMTV means mouse mammarytumor virus.

As used herein, RSV means Rous sarcoma virus; SV means Simian virus.

As used herein, CAT means chloramphenicol acetyltransferase.

As used herein, luciferase means firefly luciferase. See, de Wet, I. R.,Wood, K. V., DeLuca, M., Helinski, D. R., and Subramani, S., Mol. Cell.Biol. 7: 725-737 (1987).

As used herein, COS means monkey kidney cells which express T antigen(Tag). See Gluzman, Cell, 23:175 (1981). COS cells arereceptor-deficient cells that are useful in the functional ligandidentification assay of the present invention.

As used herein, CV-1 means mouse kidney cells from the cell linereferred to as "CV-1". CV-1 is the parental line of COS. Unlike COScells, which have been transformed to express SV40 T antigen (Tag), CV-1cells do not express T antigen. CV-1 cells are receptor-deficient cellsthat are also useful in the functional ligand identification assay ofthe present invention.

As used herein, the generic terms of art, "hormone response elements" or"HRE's", "transcriptional control units", "hormone responsivepromoter/enhancer elements", "enhancer-like DNA sequences" and "DNAsequences which mediate transcriptional stimulation", all mean the samething, namely, short cis-acting sequences (about 20 bp in size) that arerequired for hormonal (or ligand) activation of transcription. Theattachment of these elements to an otherwise hormone-nonresponsive genecauses that gene to become hormone responsive. These sequences, referredto most frequently as hormone response elements or HRE's, function in aposition- and orientation-independent fashion. Unlike other enhancers,the activity of the HRE's is dependent upon the presence or absence ofligand. (See Evans (1988) and the references cited therein.) In thepresent specification and claims, the phrase "hormone response element"is used in a generic sense to mean and embody the functionalcharacteristics implied by all terms used in the art to describe thesesequences.

As used herein, synthetic HRE's refer to HRE's that have beensynthesized in vitro using automated nucleotide synthesis machines.Since the HRE's are only about 20 bp in size, they are easilysynthesized in this manner. If wild-type, engineered or synthetic HREsare linked to hormone-nonresponsive promoters, these promoters becomehormone responsive. See Evans (1988) and the references cited therein.

As used herein, the acronym GRE means glucocorticoid response elementand TRE means thyroid receptor response element. GRE's are hormoneresponse elements that confer glucocorticoid responsiveness viainteraction with the GR. See Payvar, et al., Cell, 35:381 (1983) andSchiedereit, et al., Nature, 304:749 (1983). GRE's can be used with anywild-type or chimeric receptor whose DNA-binding domain can functionallybind (i.e., activate) with the GRE. For example, since GR, MR and PRreceptors can all activate GRE's, a GRE can be used with any wild-typeor chimeric receptor that has a GR, MR or PR-type DNA-binding domain.TRE's are similar to GRE's except that they confer thyroid hormoneresponsiveness via interaction with TR. TRE's can be used with anywild-type or chimeric receptor whose DNA-binding domain can functionallybind (i.e., activate) with the TRE. Both TR and RR receptors canactivate TRE's, so a TRE can be used with any receptor that has a TR orRR-type DNA-binding domain.

As used herein, ligand means an inducer, such as a hormone or growthsubstance. Inside a cell the ligand binds to a receptor protein, therebycreating a ligand/receptor complex, which in turn can bind to anappropriate hormone response element. Single ligands may have multiplereceptors. For example, both the T₃ R.sub.α and the T₃ R.sub.β bindthyroid hormone such as T₃.

As used herein, the word "operative", in the phrase "operative hormoneresponse element functionally linked to a ligand-responsive promoter andan operative reporter gene", means that the respective DNA sequences(represented by the terms "hormone response element", "ligand-responsivepromoter" and "reporter gene") are operational, i.e., the hormoneresponse element can bind with the DNA-binding domain of receptorprotein (either wild-type or chimeric), the ligand-responsive promotercan control transcription of the reporter gene (upon appropriateactivation by a HRE/receptor protein/ligand complex) and the reportergene is capable of being expressed in the host cell. The phrase"functionally linked" means that when the DNA segments are joined, uponappropriate activation, the reporter gene (e.g., CAT or luciferase) willbe expressed. This expression occurs as the result of the fact that the"ligand responsive promoter" (which is downstream from the hormoneresponse element, and "activated" when the HRE binds to an appropriateligand/receptor protein complex, and which, in turn then "controls"transcription of the reporter gene) was "turned on" or otherwiseactivated as a result of the binding of a ligand/receptor proteincomplex to the hormone response element.

As used herein, the phrase "DNA-binding domain" of receptors refers tothose portions of the receptor proteins (such as glucocorticoidreceptor, thyroid receptor, mineralocorticoid receptor, estrogen-relatedreceptor and retinoic acid receptor) that bind to HRE sites on thechromatin DNA. The boundaries for these DNA-binding domains have beenidentified and characterized for the steroid hormone superfamily. SeeFIG. 8; also see Giguere, et al., (1986); Hollenberg, et al., (1987);Green and Chambon (1987); and Miesfield, et al., (1987), Evans (1988).

The boundaries for the DNA-binding domains for various steroid hormonesuperfamily receptors are shown in FIG. 8-1; the boundaries are asfollows:

    ______________________________________                                        --hGR (pRShGR): nucleotide 1393 to 1590                                                       amino acid 421 to 486                                                         (See ATCC #67200)                                             --hTRb (peA101):                                                                              nucleotide 604 to 807                                                         amino acid 102 to 169                                                         (See ATCC #67244)                                             --hTRa (pherb-A8.7):                                                                          nucleotide 168 to 372                                                         amino acid 50 to 117                                                          (See ATCC #40374)                                                             amino acid 291 to 358                                                         (See ATCC #67281)                                             --ERR1 (pE4 & pHKA):                                                                          amino acid 176 to 241                                                         (See ATCC #67309 & #67310)                                    --ERR2 (phH3):  amino acid 103 to 168                                                         See ATCC #40373)                                              --hMR (pRShMR): nucleotide 2029 to 2226                                                       amino acid 603 to 668                                                         (See ATCC #67201)                                             --hRARa (phRAR1):                                                                             nucleotide 364 to 561                                                         amino acid 88 to 153                                                          (See ATCC #40392)                                             ______________________________________                                    

The DNA-binding domains of the steroid hormone superfamily of receptorsconsist of an amino segment varying between 66 to 68 amino acids inlength. The segment contains 9 cysteine residues, one of which is thefirst amino acid of the segment. This first Cys residue begins a motifdescribed as Cys-X₂ -Cys-X₁₃₋₁₅ -Cys-X₂ -Cys, where X is any amino acidresidue. The DNA-binding domain invariably ends with the amino acidsGly-Met.

For convenience in the cloning procedure, between 1 and 6 amino acidresidues preceding and/or following the DNA-binding domain can beswitched along with the DNA-binding domain.

As used herein, the phrase "ligand-binding domain region" of receptorsrefers to those portions of the receptor proteins that bind to ligandssuch as growth substances or hormones. These boundaries of theligand-binding domains for the steroid receptor superfamily have beenidentified and characterized. See FIGS. 8-1 and 8-2 and Evans (1988).

The ligand-binding domains for the various receptors are shown in FIG.8-1; some of those domains are as follows:

    ______________________________________                                        --hGR (pRShGR):    amino acid 528 to 777                                                         (See ATCC #67200)                                          --hTRb (peA101):   amino acid 232 to 456                                                         (See ATCC #67244)                                          --hTRa (pherb-A8.7):                                                                             amino acid 183 to 410                                                         (See ATCC #40374)                                          --rTR (rbeA12)     amino acid 421 to 639                                                         (See ATCC #67281)                                          --ERR1 (pE4 & pHKA):                                                                             amino acid 295 to 521                                                         (See ATCC #67309)                                          --ERR2 (phH3)      amino acid 212 to 433                                                         (See ATCC #40373)                                          --hMR (pRShMR):    amino acid 734 to 984                                                         (See ATCC #67201)                                          --hRARa (phRAR1):  amino acid 198 to 462                                                         (See ATCC #40392)                                          ______________________________________                                    

Common restriction endonuclease sites must be introduced into receptorcDNA clones to allow exchange of functional domains between receptors.In any of the various receptors referred to in FIGS. 8-1 and 8-2, thefirst common site can be introduced immediately preceding theDNA-binding domain, the second common site immediately following it.(For example, in any of the steroid hormone superfamily of receptorsthat are shown in FIGS. 8-1 and 8-2, a unique NotI site can beintroduced immediately preceding the DNA-binding domain and a uniqueXhoI site can be introduced immediately following it. This divides thereceptors into three functional regions or "cassettes"; (1) anN-terminus cassette, (2) a DNA-binding domain cassette, and (3) aligand-binding domain cassette. The three regions or cassettes from anyone receptor can be combined with cassettes from other receptors tocreate a variety of chimeric receptors.

As used herein, the nomenclature used to identify the chimeric receptorsis as follows: The various functional domains (N-terminus, DNA-bindingand ligand-binding) are identified according to the "parental" receptorfrom which they originated. For example, domains from GR are "G"domains; TR domains are "T" domains (unless otherwise further specifiedas being "T_(a) " or "T_(b) " domains); MR domains are "M" domains; RARdomains are "R" domains (unless otherwise further specified as being"R_(a) " or "R_(b) " domains), and ERR domains are "E" domains (unlessotherwise specified as being "E₁ " or "E₂ " domains). According to thisnotation, unless otherwise specified, "T" is used generically to meaneither the T₃ R.sub.α or the T₃ R.sub.β receptors; "E" means eitherhERR1 or hERR2; and "R" means either the RARα or the RARβ receptors.Wild-type receptors do not contain any exchanged domains, and soaccording to this notation system would be identified as G-G-G (or GGG),T_(a) -T_(a) -T_(a) (or T_(a) T_(a) T_(a)), T_(b) -T_(b) -T_(b) (orT_(b) T_(b) T_(b)), M-M-M (or MMM), R_(a) -R_(a) -R_(a) (or R_(a) R_(a)R_(a)), R_(b) -R_(b) -R_(b) (or R_(b) R_(b) R_(b)), E₁ -E₁ -E₁ or E₂ -E₂-E₂, where the first domain listed is the N-terminus domain, the middledomain is the DNA-binding domain, and the last domain is theligand-binding domain. Any chimeric receptor will have functionaldomains from at least two wild-type or parental sources. For example,the chimeric receptor GGR_(a) would have N-termimus and DNA-bindingdomains from glucocorticoid receptor and the ligand-binding domain fromthe alpha retinoic acid receptor; GT_(a) R_(b) would have the N-terminusfrom glucocorticoid, the DNA-binding domain from thyroid receptor alphaand the ligand-binding domain from retinoic acid receptor beta.

As used herein, hGR_(NX), hTRβ_(NX), and hRR_(NX) refer to hGR, hTRβ andhRR receptors that have been engineered to contain the unique sites forNotI and XhoI flanking the boundaries for the DNA-binding domains inthese receptors. These mutant receptors exemplify construction of hybridreceptors that are comprised of all possible combinations of aminotermini, DNA-binding domains, and ligand-binding domains from hGR, hMR,hERR1, hERR2, hTRα, hTRβ, rTRα, hRARα, and hRARβ.

As used herein, Southern blot analysis refers to a procedure fortransferring denatured DNA from an agarose gel to a nitrocellulosefilter where it can be hybridized with a complementary nucleic acid.

As used herein, Northern blot analysis refers to a technique fortransferring RNA from an agarose gel to a nitrocellulose filter on whichit can be hybridized to complementary DNA.

As used herein, "mutant" DNA of the invention refers to DNA which hasbeen genetically engineered to be different from the "wild-type" orunmodified sequence. Such genetic engineering can include the insertionof new nucleotides into wild-type sequences, deletion of nucleotidesfrom wild-type sequences, substitution of nucleotides in the wild-typesequences, or "swapping" of functional domains from one receptor toanother. Receptors that have been engineered by "swapping" functionaldomains from one receptor to another are also referred to as chimeric orhybrid receptors. Chimeric receptors can be further engineered toinclude new nucleotides, deletion of nucleotides, substitution ofnucleotides, etc.

Use of the term "substantial sequence homology" in the presentspecification and claims means it is intended that DNA, RNA, or aminoacid sequences which have slight and non-consequential sequencevariations from the actual sequences disclosed and claimed herein arewithin the scope of the appended claims. In this regard, the "slight andnon-consequential" sequence variations mean that the homologoussequences will function in substantially the same manner to producesubstantially the same compositions as the nucleic acid and amino acidcompositions disclosed and claimed herein.

As used herein, the term "recombinantly produced" means made usinggenetic engineering techniques, not merely purified from nature.

The amino acids which comprise the various amino acid sequencesappearing herein may be identified according to the followingthree-letter or one-letter abbreviations:

    ______________________________________                                                       Three-Letter                                                                             One-Letter                                          Amino Acid     Abbreviation                                                                             Abbreviation                                        ______________________________________                                        L - Alanine    Ala        A                                                   L - Arginine   Arg        R                                                   L - Asparagine Asn        N                                                   L - Aspartic Acid                                                                            Asp        D                                                   L - Cysteine   Cys        C                                                   L - Glutamine  Gln        Q                                                   L - Glutamic Acid                                                                            Glu        E                                                   L - Histidine  His        H                                                   L - Isoleucine Ile        I                                                   L - Leucine    Leu        L                                                   L - Lysine     Lys        K                                                   L - Methionine Met        M                                                   L - Phenylalanine                                                                            Phe        F                                                   L - Proline    Pro        P                                                   L - Serine     Ser        S                                                   L - Threonine  Thr        T                                                   L - Tryptophan Trp        W                                                   L - Tyrosine   Tyr        Y                                                   L - Valine     Val        V                                                   L - Glycine    Gly        G                                                   ______________________________________                                    

The nucleotides which comprise the various nucleotide sequencesappearing herein have their usual single-letter designations (A, G, T, Cor U) used routinely in the art.

As used herein, bp means base pairs and kb means kilobase pairs.

In the present specification and claims, the Greek letters alpha (α),beta (β), etc. are sometimes referred to as a, b, etc.

DEPOSITS

Plasmids pRShGR (hGR), pRShMR (hMR), peA101 (hT₃ β) and GMCAT, all ofwhich are in E. coli HB101, plus plasmids rebA12 (rTRα), pE4 and phKA(which together encode hERR1), phH3 (hERR2), pherb-A 8.7 (hTRα), phFA 8(a partial clone of hTRα), and plasmid phRARI have been deposited at theAmerican Type Culture Collection, Rockville, Md., U.S.A. (ATCC) underthe terms of the Budapest Treaty on the International Recognition ofDeposits of Microorganisms for Purposes of Patent Procedure and theRegulations promulgated under this Treaty. Samples of the plasmids areand will be available to industrial property offices and other personslegally entitled to receive them under the terms of said Treaty andRegulations and otherwise in compliance with the patent laws andregulations of the United States of America and all other nations orinternational organizations in which this application, or an applicationclaiming priority of this application, is filed or in which any patentgranted on any such application is granted.

The ATCC Deposit Numbers and Deposit Dates for the deposits are asfollows:

    ______________________________________                                        pRShGR (hGR)     67200     Sept. 9, 1986                                      pRShMR (hMR)     67201     Sept. 9, 1986                                      pE4 (hERR1*)     67309     Jan. 30, 1987                                      phHKA (hERR1*)   67310     Jan. 30, 1987                                      phH3 (hERR2)     40373     Sept. 29, 1987                                     GMCAT (reporter) 67282     Dec. 18, 1986                                      pherb-A 8.7 (hTRa)                                                                             40374     Sept. 29, 1987                                     phFA 8 (hTRa*)   40372     Sept. 29, 1987                                     peA101 (hTRb)    67244     Oct. 22, 1986                                      prbeA12 (rTRa)   67281     Dec. 18, 1986                                      phRARa (hRARa)   40392     Nov. 20, 1987                                      ______________________________________                                         (*means a partial clone)                                                      (pE4 & phHKA together encode complete hERR1)                             

SUMMARY OF THE INVENTION

In one aspect, the present invention comprises a double-stranded DNAsegment wherein the plus or sense strand of the segment contains asequence of triplets coding for the primary sequence of a protein whichhas ligand-binding and DNA-binding (or transcription-activating)properties characteristic of a retinoid receptor protein referred toherein as human retinoic acid receptor protein. According to this aspectof the invention, the double-stranded DNA segment is one which iscapable of being expressed into retinoic acid receptor protein.

In another aspect, the invention comprises a single-stranded DNA, whichis the sense strand of a double-stranded DNA coding for retinoic acidreceptor protein, and an RNA made by transcription of thisdouble-stranded DNA.

In another aspect, the invention comprises a plasmid, phRARI, whichcontains DNA coding for a retinoic acid receptor protein of the presentinvention (RARα). This plasmid has been deposited with the American TypeCulture Collection for patent purposes; it has been accorded ATCC No.40392.

In still another aspect, the invention comprises a cell, preferably amammalian cell, transformed with a DNA coding for retinoic acid receptorprotein. According to this aspect of the invention, the transforming DNAis capable of being expressed in the cell, thereby increasing the amountof retinoic acid receptor, encoded by this DNA, in the cell.

Further the invention comprises novel retinoic acid receptors made byexpression of a DNA coding for retinoic acid receptor or translation ofan mRNA transcribed from such a retinoic acid receptor coding DNA.According to this aspect of the invention, the retinoic acid receptorswill be protein products of "unmodified" retinoic acid coding DNA's andmRNA's, or will be modified or genetically engineered retinoic acidreceptor protein products which, as a result of engineered mutations inthe receptor DNA sequences, will have one or more differences in aminoacid sequence from the corresponding naturally occurring "wild-type"retinoic acid receptor proteins. Preferably these retinoic acidreceptors, whether "unmodified" or "engineered", will have at leastabout 5% of the retinoic acid binding activity and/or at least about 5%of the DNA-binding or transcription-activating activity of thecorresponding naturally occurring retinoic acid receptor.

Further the invention comprises chimeric receptors made by exchangingthe functional domains of one receptor with functional domains ofanother type. The chimeric DNA's thus produced encode chimeric receptorproteins that have functional characteristics based on the "origin" oftheir respective DNA- and ligand-binding domains. The chimeric receptorsof the invention include double-stranded DNA's that code for thechimeric receptors, as well as single-stranded DNA's which are the sensestrands of the double-stranded DNA's, and mRNA's made by transcriptionof the double-stranded DNA's. The invention also comprises cells, botheukaryotic and prokaryotic, that are transformed with chimeric receptorsencoding DNA's of the invention.

According to the chimeric receptor aspect of the invention, to effectthe chimeric DNA fusions, two restriction endonuclease sites areintroduced into each receptor cDNA at comparable locations in or nearthe DNA-binding domains in order to divide the receptor DNA's into threefunctional domains or regions. (For example, a unique NotI site can beintroduced immediately preceding the DNA-binding domain and a uniqueXhoI site can be introduced immediately following it. This divides thereceptors into three functional regions or "cassettes"; (1) anN-terminus cassette, (2) a DNA-binding domain cassette, and (3) aligand-binding domain cassette. The three regions or cassettes from anyone receptor can be combined with cassettes from other receptors tocreate a variety of chimeric receptors. This aspect of the invention isillustrated in the section of the specification labeled "DetailedDescription of the Invention".)

In the present specification and claims, the chimeric receptors(referred to also as chimera or hybrids) are named by letters referringto the origin of the various domains. Domains from hGR are referred toas "G" domains, domains from hTR are "T" domains, domains from hERR are"E" and domains from hRR are "R" domains. For example, the chimericreceptor "RGR" has the amino and carboxyl termini of hRR and theDNA-binding domain of hGR; the chimeric receptor "TGG" has the aminoterminus from hTR, and the DNA-binding and carboxyl terminus from hGR.(In the diagram shown in FIG. 7, the amino terminus of the receptor isreferred to domain A/B and the carboxyl terminus is referred to asdomain E.)

According to the notation used in the specification and claims, unlessotherwise specified, "T" is used generically to mean either the T₃R.sub.α or the T₃ R.sub.β receptor; "E" means either hERR1 or hERR2; and"R" means either the RARα or the RAPβ receptor.

Chimeric receptors of the invention include chimera having (1) anN-terminus domain selected from the group of wild-type receptorsconsisting of hGR, hMR, hERR₁, hERR₂, rTRα, hT₃ α, hT₃ β, hRARα andhRARβ, (2) a DNA-binding domain selected from the group of wild-typereceptors consisting of hGR, hMR, hERR₁, hERR₂, rTRα, hT₃ α, hT₃ β,hRARα and hRARβ, and (3) a ligand-binding domain selected from the groupof wild-type receptors consisting of hGR, hMR, hERR₁, hERR₂, rTRα, hT₃α, hT₃ β, hRARα and hRARβ, wherein any one chimeric receptors will haveN-terminus, DNA-binding, and ligand-binding domains that originate fromat least two different "wild-type receptor" sources.

Preferred chimeric receptor DNA's of the invention include GRR, GRG,GGR, RGG, RGR, RRG, GTT, GTG, GGT, TGG, TGT, TTG, TTR, TRT, TRR, RTT,RTR, RRT, GTT, GTG, GGT, TGG, TGT, and TTG receptor DNA's, plus thechimeric hybrid receptor proteins made by expression of a chimeric DNAof the invention or translation of an mRNA transcribed from such achimeric receptor coding DNA. Preferably these chimeric receptors willhave activity that exceeds exogenous background binding ortranscriptional activation activity levels in any given cell, or willhave at least about 5% of the DNA-binding or transcription-activatingactivity of the corresponding naturally occurring receptor DNA-bindingdomain, and/or about 5% of the ligand-binding activity of thecorresponding naturally occurring ligand-binding domain.

The invention also comprises a method for identifying functionalligand(s) for receptor proteins. According to the method, DNA sequences(referred to herein as the sample sequences) can be isolated which codefor receptor proteins and which have at least an operative portion of aligand-binding domain and a DNA-binding domain. (As those skilled in theart will appreciate, not all of the DNA sequences in the ligand-bindingdomains are necessary in order for the domains to be functional. Theoperative sequences, i.e., those that must be present if the domain isto bind ligand, can be identified by deletion studies on any givendomain.) Once the sample DNA sequences are isolated, a chimeric gene canbe created by substituting the DNA-binding domain region in the sampleDNA sequence with a DNA-binding domain region taken from a DNA sequencecoding for another receptor protein, e.g., glucocorticoid receptorprotein, thyroid receptor protein, mineralocorticoid receptor protein orretinoic acid receptor protein. Next a suitable receptor-deficient hostcell is transfected with: (1) the chimeric receptor gene, which ispreferably carried on an expression plasmid, and (2) a reporter gene,such as the CAT gene or the firefly luciferase gene, which is alsopreferably carried on plasmid, and which is referred to in U.S. Ser. No.108,471 as a reporter plasmid. In any case, the reporter gene isfunctionally linked to an operative hormone response element (HRE)(either wild-type or engineered) wherein the hormone response element iscapable of being activated by the DNA-binding domain used to make thechimeric receptor gene. (For example, if the chimeric receptor genecontains the DNA-binding domain region from glucocorticoid receptorcoding DNA, then the HRE should be a wild-type, an engineered, or asynthetic GRE, i.e., one that can be activated by the operative portionof the DNA-binding region of a glucocorticoid receptor protein. If athyroid receptor DNA-binding domain region is used, then the wild-typeor engineered HRE should be responsive to a thyroid (or retinoic acid)receptor protein, etc.) Next the transfected host cell is challengedwith a battery of candidate ligands which can potentially bind with theligand-binding domain region of the chimeric protein coded for by thechimeric gene. To determine which of these ligands can functionallycomplex with the chimeric receptor protein, induction of the reportergene is monitored by monitoring changes in the protein levels of theprotein coded for by the reporter gene. (For example, if luciferase isthe reporter gene, the production of luciferase is indicative ofreceptor-regulated gene transcription.) Finally, when a ligand(s) isfound that can induce transcription of the reporter gene, it isconcluded that this ligand(s) can bind to the receptor protein coded forby the initial sample DNA sequence. This conclusion can be furtherverified by testing the binding properties of the receptor protein,coded for by the initial sample DNA sequences, vis-a-vis the ligand(s)that induce expression of the reporter gene.

As those skilled in the art will appreciate, if a cell already contains(a) a chimeric DNA sequence (C) comprised of (1) operative portions of aDNA-binding domain of a first receptor sequence (i.e., a first sequence)linked to (2) operative portions of a ligand-binding domain of a secondreceptor sequence (i.e., a second sequence), and (b) a reporter nucleicacid sequence functionally linked to an operative hormone responseelement wherein the operative portions of the DNA-binding domain of thefirst receptor sequence can functionally bind to and activate thehormone response element that is functionally linked to the reportersequence, then the method for identifying a functional ligand for areceptor protein will be comprised of challenging the cell with at leastone candidate ligand and then monitoring induction of the reportersequence by means of changes in the amount of expression product of thereporter sequence.

The new functional ligand identification assay makes it possible toscreen a large number of potential ligands or any given receptors,regardless of whether the receptor is a wild-type receptor or a chimericone.

The functional ligand identification method is illustrated herein byshowing (1) that the retinoid, retinoic acid and its metabolicprecurser, retinol, are functional ligands for the receptor proteincoded for by phRARl DNA, and (2) that the DNA- and ligand-bindingdomains determine the functional characteristics of the chimericreceptors.

The new functional assay, as well as the new retinoic acid receptor andthe new chimeric receptors, are described more fully below.

DESCRIPTION OF THE INVENTION The Retinoic Acid Receptor

In a continuing effort to explore the steroid hormone receptorsuperfamily, advantage was taken of the fortuitous identification of anovel genomic sequence with striking homology to the DNA-binding domainof the steroid hormone receptors (see Dejean et al., 1986). Thissequence spans the integration site of a hepatitis B virus (HBV) from ahuman hepatocellular carcinoma.

To pursue the hypothesis that this gene might code for a previouslyunknown receptor, an oligonucleotide derived from this sequence waslabeled and used to probe a number of human cDNA libraries. Fivepositive clones were initially isolated from a testis cDNA library. Theinsert from one of these clones (1hT1R) was used to isolate additionalcDNA clones from a λgt10 kidney cDNA library. A restriction map of thelargest clone (phRARl) is shown in FIG. 1A. Nucleotide sequence analysisreveals a long open reading frame of 462 amino acids beginning with apresumptive initiator methionine codon corresponding to nucleotides103-105 as shown in FIG. 1B-1. The sequence surrounding this ATG agreeswith the consensus described by Kozak (1987) for a translationinitiation site. Upstream of the ATG is an in-frame terminator providingsupport for the initiator methionine. Another methionine found 30 codonsdownstream fails to conform to the consensus and is an unlikelyinitiator. Following the terminator codon at position 1489-1491 is a3'-untranslated region with a consensus polyadenylation signal (AATAAA)found 20 nucleotides upstream of a polylated tract (see Proudfoot, etal., 1976).

A polypeptide of relative molecular mass 50,772 d (51 Kd) is encodedwithin the translational open reading frame. The size of the proteinencoded by the insert of phRARI was verified by in vitro translation ofRNA (see Krieg, et al., (1984)) derived from this insert and found tocorrespond to the predicted size of 54 Kd (data not shown). Amino acidsequence of this protein has been compared to the glucocorticoid andthyroid hormone receptors. The highest degree of similarity is found ina cysteine-rich sequence of 66 amino acids beginning at residue 88. Ourgroup has previously demonstrated that this region of the hGR representsthe DNA-binding domain for this receptor. See Giguere, et al., (1987)and Hollenberg, et al., (1987). In addition, mutagenesis and expressionstudies have provided direct evidence for its role in transcriptionalactivation of genes harboring glucocorticoid response elements (GREs).See Giguere, et al., (1987) and Hollenberg, et al., (1987).

Domain Switching and Transcriptional Activation

Since the ligand for the gene product of phRARI was unknown, it wasdesirable to develop a quick and sensitive assay to reveal its identity.Previous studies have demonstrated that the DNA-binding domain of thehuman glucocorticoid and estrogen receptors can be interchanged to yielda functional hybrid receptor. This chimera recognizes the glucocorticoidresponsive element of the MMTV-LTR but stimulates transcription in anestrogen-dependent fashion (see Green, et al., (1987)). This led us towonder if a general domain-swapping strategy could be exploited toidentify the ligand-binding properties of a novel hormone receptor. Totest this approach we first substituted the DNA-binding domain of thephRARI gene product with the well described DNA-binding domain from thehGR (FIG. 2A). (This chimeric construction, when expressed in suitablehost cells, produces a hybrid receptor protein whose ligand-bindingdomain region must bind with a functional exogenous ligand before theligand/receptor complex can bind to a GRE, thereby activating aglucocorticoid inducible promoter.)

To assay for the presence of a functional ligand the chimeric receptorgene was transfected into suitable host cells along with a suitable GRElinked reporter gene. CV-1 cells were used for the assay along with aMMTV-CAT reporter gene. (MMTV-CAT is carried on reporter plasmid,GM-CAT, which has been deposited with the American Type CultureCollection for patent purposes; see the section of this specificationlabeled, "Deposits". As those skilled in the art will appreciate,reporter plasmids suitable for assaying hybrid thyroid receptorproteins, i.e., hybrid proteins having the DNA-binding domain of athyroid receptor protein, can be constructed by substituting the GRE onplasmid GM-CAT with a thyroid hormone responsive transcription element.For example, the growth hormone promoter can be functionally linked tothe bacterial CAT gene. Since the growth hormone promoter contains athyroid responsive transcription element, such a reporter plasmid can beused to assay hybrid thyroid receptor proteins. See the subheading:"Construction of Reporter and Expression Plasmids" in thisspecification. (Since mineralocorticoid receptors can activate GRE's, areporter plasmid such as GM-CAT can be used to assay hybridmineralocorticoid receptor proteins.)

Returning to the functional ligand identification assay, the transfectedcells were then systematically challenged with a battery of candidateligands and induction monitored by changes in CAT activity.

Because of their hormonal-like activities, the retinoids, includingretinol (Vitamin A) and retinoic acid, were evaluated as potentialinducers. Remarkably, retinoic acid elicited a dramatic increase in CATactivity of the hybrid receptor (FIG. 2B). No effect upon CAT activitywas observed using the parent vector, pRShRR_(NX), or the wild type geneproduct from phRAR1, herein referred to as human retinoic acid receptor(hRARα). As expected, the hybrid receptor is not induced byglucocorticoids, and the hGR is not induced by retinoic acid.

As shown in FIG. 3A, retinoic acid exhibits an ED₅₀ value of 6×10⁻¹⁰ Mon CAT activity induced by the hybrid receptor, which is consistent withED₅₀ values observed for retinoic acid in a variety of biological assays(see Sporn and Roberts, 1984). Retinol functions as a weak agonist withan ED₅₀ value greater than 100 nM. Retinyl acetate and retinyl palmitatefunction as even weaker inducers. A number of natural and syntheticligands including testosterone, dihydrotestosterone, estrogen,dexamethasone, cortisol, aldosterone, progesterone, T₃, T₄, Vitamin D₃and 25-OH-cholesterol failed to induce CAT activity.

To corroborate the identity of the phRARI gene product as the retinoicacid receptor, the binding properties of the expressed product wereevaluated following transfection of COS-1 cells. As shown in FIG. 3B,transfected cells reveal increased capacity to specifically bind ³H-retinoic acid. This increase occurs over an endogenous background thatis a likely consequence of the presence of cellular retinoid bindingproteins as well as a significant non-specific binding. Consistent withthe activation studies, the binding is fully competed by retinoic acidbut only partially by retinol. Thyroid hormones, dexamethasone andvitamin D₃ did not compete the binding of retinoic acid.

A Gene Family

To determine if the new retinoic acid gene was unique and to identifypotentially related genes, human DNA was examined by Southern blotanalysis. Hybridization of restriction endonuclease-digested human DNAwith a labeled DNA fragment derived from the coding region of the hRRgene produced three bands in every digestion consistent with a singlehybridizing genetic locus (FIG. 4A). This hybridization pattern isunrelated to the restriction endonuclease map described by Dejean et al.(1986) for the HBV pre-integration site. However, when the hybridizationconditions were relaxed, six additional bands were observed in theproducts of each enzyme digestion (FIG. 4B). These observationssuggested that there were at least one additional locus, and possiblymore, in the human genome related to the retinoic acid receptor. TheRARβ has now been found. See Brand, et al, (1988).

Expression of the hRR Gene

Since retinoic acid is known to exert effects on a large number ofdifferent cell types, we examined the expression of the hRR gene. Totalcytoplasmic RNAs isolated from a variety of rat and human tissues weresize fractionated and transferred to a nitrocellulose filter.Hybridization with a 600-bp restriction fragment from phRAR1 reveals amajor RNA species of 3,200 nucleotides with highest levels in thehippocampus, adrenals, cerebellum, hypothalamus and testis (FIG. 5).Longer exposure shows that most tissues contain a small amount of the3.2 kb transcript while it is undetectable in some tissues such asliver.

Retinoic Acid Receptor Data Summary

The data disclosed herein identify the gene product of phRAR1 as a humanretinoic acid receptor based on three criteria. First, the overallstructural homology of the hRR to steroid and thyroid hormone receptors(FIG. 6) suggests that it is likely to be a ligand-responsive regulatoryprotein. Second, an expressed chimeric receptor, consisting of theDNA-binding domain of the hGR and the presumptive ligand-binding domainof the hRR acts as a transcriptional regulator of aglucocorticoid-inducible reporter gene only in the presence of retinoicacid. This induction occurs at physiological levels. Third, expressionof the candidate hRR in transfected cells selectively increases thecapacity of those cells to bind retinoic acid.

Development and Oncogenesis

The retinoids comprise a group of compounds including retinoic acid,retinol (vitamin A) and a series of natural and synthetic derivativesthat together exert profound effects on development and differentiationin a wide variety of systems. See Sporn & Roberts, (1983); Mandel &Cohen, (1985); Wolback & Howe, (1925); Lotan (1980); and Fuchs & Green,(1980). Although early studies focused on the effects of retinoids onepithelial growth and differentiation, their actions have been shown tobe more widespread than previously suspected. Many recent studiesdemonstrate the effects of these molecules on a variety of cultured celllines including neuroblastomas (see Hausler, et al., (1983)), melanomas(see Lotan, et al., (1983)) and fibroblasts (see Shroder et al.,(1982)). In the human promyelocytic leukemia cells (HL-60), retinoicacid is a potent inducer of granulocyte differentiation (see Breitman,et al., (1980)). In F9 teracarcinoma stem cells, retinoic acid willinduce the differentiation of parietal endoderm, characteristic of alate mouse blastocyst (see Strickland & Mahdavi, (1978); Jetten et al.,(1979); and Wang et al., (1985)). Retinoic acid has been shown to exertequally potent effects in development. For example, in the developingchick limb bud, retinoic acid is able to substitute for the action ofthe polarizing region in establishing the anterior-posterior axis (seeTickle & Eichele, (1985)). By controlling the exposure to retinoic acid,it is possible to generate novel patterns of limb structures. Althoughretinoic acid is primarily considered a morphogen, Northern blotanalysis suggests a re-evaluation of its function in the adult. Inhumans, retinol deficiency has been linked to an alarming increase in avariety of cancers (see Moon & Itri, (1984)). Retinoids have also beenshown to inhibit tumor progression in animals and block the action oftumor promoters in vitro. In this context, the hRR may be considered asa negative regulator of oncogenesis.

A Superfamily of Regulatory Genes

Two surprising results have emerged from the studies presented here. Thefirst is the discovery of a family of retinoic acid receptor-relatedgenes which predicts the existence of one or more other proteins withclosely related properties (e.g., the RARβ described by Brand et al.,(1988)). Physiological studies demonstrate that both retinoic acid aswell as retinol (vitamin A) can exert potent effects on cellulardifferentiation and that these effects are often not linked. It thusseems likely that at least one related gene product might be a specificretinol receptor or a receptor for another member of the retinoidfamily. The second surprising observation from these results is theclose kinship of the retinoid receptor with the thyroid hormonereceptor. (As we show below, the retinoic acid receptor can activate athyroid response element or TRE; see the section of the specificationlabeled "Retinoic Acid and Thyroid Hormone Induce Gene ExpressionThrough a Common Response Element".) This relationship is surprising inpart because of the structural dissimilarity of the thyroid hormones andthe retinoids. Thyroid hormones being derived from the condensation oftwo tyrosine molecules whereas, the retinoids are derived from mevalonicacid. The observation that chemically distinct molecules interact withreceptors sharing common structures most likely reflects a common modeof action with which they elicit their particular regulatory effects.Based on this analogy, we can now propose that the interaction ofretinoids with their intracellular receptors induces a cascade ofregulatory events that results from the activation of specific sets ofgenes by the hormone/receptor complex. Although animals employ diversemeans to control their development and physiology, the demonstrationthat the retinoic acid receptor is part of the steroid receptorsuperfamily suggests that mechanisms controlling morphogenesis andhomeostasis may be more universal than previously suspected.

Construction and Characterization of Chimeric Receptors

Construction of chimeric receptor genes is discussed above in thesections of the specification labeled "Definitions", "Summary of theInvention" and "Domain Switching and Transcriptional Activation". In thesections that follow, construction and characterization of the chimericreceptors is illustrated by showing construction and characterization ofGR/TR hybrids.

Materials and Methods Cell Culture and Transfection

CV-1 cells were used as the receptor-deficient host cells that weretransfected with expression plasmids that carry the chimeric RR/TRreceptors, and reporter plasmids carrying the CAT reporter gene.Conditions for growth and transfection of CV-1 (African Green monkeykidney) cells were as previously described (Giguere et al. (1986)),except that the calcium phosphate precipitate was left on the cells for4-8 hours, at which time the media was changed to DMEM with 5% T₃ freebovine serum (Scantibodies) minus or plus 10⁻⁷ M T₃ (Sigma). Cells wereharvested 36 hours after the addition of T₃, and CAT assays wereperformed as described by Gorman et al. (1982). Typically, 5 μg reporterand 1 μg expression vector were cotransfected, along with 2.5 μgRSV-βgal as a control for transfection efficiency. Acetylated andnon-acetylated forms of [¹⁴ C]chloramphenicol were separated by thinlayer chromatography, excised, and quantitated by liquid scintillationcounting in Econofluor (DuPont) with 5% DMSO. β-galactosidase assayswere performed as described by Herbomel et al. (1984). CAT activity isexpressed as percent conversion divided by β-galactosidase activity.

Construction of Reporter and Expression Plasmids

Synthetic oligonucleotides corresponding to -169 to -200 of the ratgrowth hormone gene was inserted into a linker scanning mutant of MTV-CAT that has a HindIII site at position -190/-181 (Buetti and Kuhnel(1986)). Expression vectors were constructed for the thyroid hormonereceptors by inserting the full-length cDNAs of pheA12 (hTRβ, seeWeinberger, et al. (1986)) and rbeA12 (rTRα, see Thompson, et al.(1987)) between the KpnI and BamHI sites of the pRS vector (Giguere, etal. (1986) and (1987)).

Construction of Chimeric Receptors

The construction of hGR_(NX) has been described (Giguere, et al. (1987).To construct hTRβ_(NX), the cDNA insert of pheA12 (hTRβ, see,Weinberger, et al. (1985) and (1986)) was subcloned between the KpnI andBamHI sites of M13mp19 and mutagenized by the method of Kunkel (1985).The oligonucleotide used to create the NotI site changed three aminoacids: Asp97 to Arg, Lys98 to Pro, Asp99 to Pro. The oligonucleotideused to create the XhoI site changed two amino acids: Thr171 to Leu,Asp172 to Gly. The mutant receptor cDNA was then transferred to theexpression vector pRS (Giguere, et al. (1986) and (1987)); hybrids wereconstructed by exchanging KpnI-NotI, KpnI-XhoI, or NotI-XhoI restrictionfragments between RShGR_(NX) and RShTRβ_(NX). RShGR_(NX) has about 75%of wild-type DNA-binding activity, and RShTRβ_(Nx) has about 60% ofwild-type DNA-binding activity.

The Cis/Trans Functional Ligand Identification Assay

The cis/trans functional ligand identification cotransfection assay wasused to study chimeric receptors constructed by swapping domains betweenthe glucocorticlod, the thyroid and the retinoic acid receptors. (Asthose skilled in the art will appreciate, the cis/trans cotransfectionassay can be used to study chimeric receptors made by swappingfunctional between any of the wild-type or genetically engineeredreceptors.) In the cis/trans assay, preferably two plasmids aretransfected into a receptor deficient cell line. The first plasmid isused to express the receptor protein (whether wild-type, chimeric orgenetically engineered). The second plasmid is used to monitortranscription from a ligand or hormone responsive promoter. For thethyroid hormone receptor assay, the expression plasmid consists of theRous Sarcoma Virus long terminal repeat (RSV-LTR) directing theexpression of a cDNA encoding a thyroid hormone receptor. For the hGR,the reporter plasmid is the mouse mammary tumor virus long terminalrepeat (MTV-LTR) fused to the bacterial chloramphenicolacetyltransferase (CAT) gene. To convert MTV-CAT to a thyroid hormoneresponsive reporter, an oligonucleotide containing a thyroid hormoneresponse element (TRE) was inserted at position -191 of the MTV-LTR.This sequence, -169 to -200 of the rat growth hormone (rGH) gene,specifically binds thyroid hormone receptors and can confer T₃responsiveness to a heterologous promoter (Glass et al. (1987)).Expression and reporter plasmids were cotransfected into CV-1 cells andCAT activity was measured in the absence and presence of T₃. The assaysshowed that neither the alpha nor the beta thyroid hormone receptoractivates transcription from MTV-CAT, in the absence or presence of T₃.(Data not shown.) However, the addition of a TRE produces an MTVpromoter that is thyroid hormone responsive. Induction of CAT activityis dependent on the cotransfection of a functional alpha or beta thyroidhormone receptor and the addition of T₃. In the presence of T₃, thealpha receptor (rTRα) induces CAT activity approximately 15-fold, whilethe beta receptor (hTRβ) induces activity by about 5-fold.

The hybrid thyroid hormone/glucocorticoid receptors were constructed tocompare the functional properties of the thyroid and glucocorticoidhormone receptors. To facilitate the construction of the chimeric hybridreceptors, unique sites for the restriction enzymes NotI and XhoI wereinserted flanking the DNA binding domains of hGR and hTRβ. These mutantreceptors, termed hGR_(NX) and hTRβ_(NX), can be used to create hybridswith all possible combinations of amino termini, DNA-binding domains,and ligand-binding domains for these receptors. (As those skilled in theart will appreciate, comparable plasmids, such as pRARα_(NX) or pMR_(NX)for example, can be used to create chimeric receptors consisting of allpossible combinations of all functional domains from the variousreceptors in the steroid hormone receptor superfamily. The receptors andthe locations of the various functional domains are shown in FIG. 8-1.)The hybrid and parental receptors were assayed using both thyroidhormone and glucocorticoid responsive promoters, in the absence orpresence of T₃ or the synthetic glucocorticoid dexamethasone.

The structures and activities of the hybrid thyroid/glucocorticoidreceptors are shown in FIG. 9. The receptors are divided into threesections, and hybrids are named by letters referring to the "origin" ofthe domain; for example, "T-G-T" has the amino and carboxyl termini ofhTRβ (T-, -T) and the DNA binding domain of the hGR (-G-). Hybrids witha putative hTRβ DNA binding domain (TTG, GTT, GTG) activatedtranscription from TRE- CAT, while hybrids with an hGR DNA bindingdomain (GGT, TGG, TGT) activated transcription from GRE- CAT. Thisdemonstrates that this region of hTRβ is analogous to the hGR DNAbinding domain and is responsible for promoter recognition. Hybridreceptors with an hTRβ carboxyl terminus were activated by T₃, whilethose with an hGR carboxyl terminus were activated by dexamethasone.This is consistent with the identification of the carboxyl terminus asthe part of the receptor that is responsible for hormone binding andactivation specificity. Taken together, the functional properties ofthese hybrids support the assignment of the DNA- and ligand-bindingdomains of hTRβ.

Retinoic Acid and Thyroid Hormone Induce Gene Expression Through aCommon Responsive Element

Identification of a functional retinoic acid responsive element (RARE)is crucial to our understanding of the mechanisms by which retinoic acidreceptors activate gene expression and regulate cell differentiation.One impediment to such a study is the absence of any identified genewhose transcription is directly dependent on the retinoic acidreceptor-hormone complex. An alternative approach to localize a RARE isto systematically challenge the inducibility of known hormonallyresponsive promoters with retinoic acid receptor produced from clonedcDNA. (As discussed above under the heading "The Cis/Trans Assay", inthis system, transcriptional activation from a promoter containing a HREis dependent on expression of functional receptor from cotransfectedexpression plasmids in receptorless cells such as CV-1.) Because theDNA-binding domains of the retinoic acid and thyroid hormone receptorsare highly related (62% identical in their amino acid sequences, seeFIG. 6), the possibility that the retinoic acid receptor could activategene expression through a TRE was investigated.

TRE's are known; see, for example, Glass, et al., (1987) for adiscussion of a cis-acting element in the rat growth hormone 5' flankinggenomic sequence that is necessary for thyroid hormone(3,5,3'-triiodo-L-thyronine, T₃) regulation.

To test if a TRE could effectively function as a RARE, a novel T₃responsive promoter was constructed by replacing the glucocorticoidresponsive elements present in the mouse Mammary Tumour Virus-LongTerminal Repeat (MTV-LTR) with an oligonucleotide encoding the naturalTRE_(GH). This promoter was then fused to the bacterial chloramphenicolacetyl transferase (CAT) gene to generate the reporter plasmidΔMTV-TRE_(GH) -CAT. After transient transfection into CV-1 cells, theinducibility of the promoter was determined by measuring CAT activity.When CV-1 cells are cotransfectd with the expression vector containing ahuman thyroid hormone receptor beta (pRShT₃ Rβ) and the reporter plasmidΔMTV-TRE_(GH) -CAT, induction in CAT activity is observed in thepresence of T₃. In contrast, cotransfection of an expression vectorencoding the human glucocorticoid receptor (pRShGRα) and the samereporter plasmid did not stimulate CAT activity from this promoter inresponse to the synthetic glucocorticoid dexamethasone. These resultsclearly demonstrate that the induction of CAT activity by RARα isconferred by the TRE because the wild-type MTV-LTR construct was notresponsive. (Data not shown.) These results also show that the hRARα canspecifically induce gene expression from a promoter containing a TRE.

RAR and GR Chimeric Receptors

As discussed above, the modular structure of steroid hormone receptorsmakes it possible to exchange functional domains from one receptor toanother to create functional chimeric receptors. This strategy was usedto create hGR/hRARα chimera that had the RAR DNA-binding domain and theGR ligand-binding domain. When CV-1 cells were cotransfected with theexpression plasmid encoding hGRG and the reporter ΔMTV-TRE_(GH) -CAT,dexamethasone specifically elicited CAT activity. (Data not shown.) Thisexperiment provided direct evidence that the DNA-binding domain of thehRARα determined the specificity of target gene activation.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1. DNA and primary amino acid sequence of phRAR1. A, Schematicrepresentation and restriction enzyme map of the phRAR1 clone. Thestippled box represents the predicted open reading frame. B, (shown asB-1, B-2 and B-3) The complete nucleotide sequence of phRAR1 is shownwith the amino acid sequence given above the long open reading frame. Anupstream in-frame stop codon at nucleotides 85-87 and polyadenylationsignal are underlined.

FIG. 1 Methods. A 63-mer oligonucleotide corresponding to nucleotides408-477 of the genomic sequence published by Dejean et al. (1986) wasused as a hybridization probe to screen a human testis λgt10 library.The hybridization mixture contained 35% formamide, 1× Denhardt's, 5×SSPE, 0.1% sodium dodecyl sulfate (SDS), 100 μg ml⁻¹ denaturated salmonsperm DNA and 10⁶ c.p.m. ml⁻¹ of ³² P-labeled oligonucleotide. Duplicatenitrocellulose filters were hybridized at 42° C. for 16 h, washed threetimes for 20 min each in 2× SSC, 0.1% SDS (1×SSC=150 mM NaCl, 15 mMsodium citrate) at 55° C. and autoradiographed at -70° C. with anintensifying screen. Clone1hT1R obtained from this screening waspartially characterized and then used as a hybridizing probe to screen ahuman kidney λgt10 cDNA library (see Bell, et al., (1986)). For thisscreening, the washing conditions were modified to 1×SSC with 0.1% SDSat 68° C. Several cDNA clones were isolated and the longest clone,phRAR1, was digested with a number of restriction enzymes and theresulting fragments were subcloned in both orientations into the M13sequencing vectors mp18 and mp19 and sequenced by the dideoxy procedure(see Sanger, et al., (1977)). DNA sequences were compiled and analyzedby the programs of Devereux et al. (1984) and Staden (1982).

FIG. 2. A, Construction of the chimeric receptor hRGR. Thedomain-structure of the various constructions are shown schematically,the numbers correspond to the amino acid positions of each domain. TheDNA-binding domains are represented by "DNA" and the ligand-bindingdomains by their respective inducers. The Not1 and Xho1 sites created bysite-directed mutagenesis to permit the exchange of the DNA-bindingdomains between receptors are indicated. B, Induction of CAT activity byretinoic acid. The expression vectors were cotransfected into CV-1 cellswith the reporter plasmid MTVCAT and cultured for 2 days in absence orpresence of 100 nM dexamethasone (DEX) or retinoic acid (RA). Thereceptor inserted into the expression vectors are: pRShGR, humanglucocorticoid receptor; pRShRR, human retinoic acid receptor;pRShRR_(nx), mutated human retinoic acid receptor with Not1 and Xho1sites; pRShRGR, chimeric receptor composed of the human retinoic acidreceptor which DNA-binding domain has been replaced by the humanglucocorticoid receptor DNA-binding domain.

FIG. 2 Methods. A, Restriction enzyme fragments of the cDNA inserts ofphRAR1 and hGR (see Hollenberg, et al., (1985) were subcloned into theKpn1 and BamH1 sites of the mp19 vector and mutagenized according to themethod of Kunkel (1985). The oligonucleotides used for the creation ofthe Not1 site within hGR and hRR were 28 and 31 nucleotidesrespectively, while the oligonucleotides used for the creation of theXho1 site within hGR and hRR were 24 and 23 nucleotides. The creation ofthe Not1 site resulted in the mutation of Pro₄₁₆ to an Arg residue inhGR_(NX), and in the mutation of Ile₈₄ and Tyr₈₅ to Pro residues inhRR_(NX). The introduction of the Xho1 site did not alter the hGR_(NX)amino acid sequence but resulted in the mutation of Lys₁₅₅ to a Leuresidue in hRR_(NX). The mutant receptors were then transferred to theexpression vector pRS (see Giguere, et al., (1986), and the Not1/Xho1restriction fragment of pRShGR_(NX) containing the hGR DNA-bindingdomain was introduced into pRShRnx between the Not 1 and Xho1 sites tocreate pRShRGR. B, Cell transfection and CAT assay. The recombinant DNAconstructs (5 μg each) were introduced into CV-1 cells by calciumphosphate coprecipitation (see Wigler, et al., (1979)). The cells werethen cultured for two days in serum free media supplemented withNutridoma (Boehringer Mannheim) in presence or absence of inducers. CV-1cells were then prepared for CAT assays as described by Gorman, et al.(1982) and the assays performed for 3 h using 25 μg of protein extract.All experiments with retinol were conducted in subdued light.

FIG. 3. A, Dose-response to retinoids. CV-1 cells cotransfected withpRShRGR and pMTVCAT were treated with increasing concentrations ofretinoids or a single 1 μM dose (*) of testosterone,dihydrotestosterone, estrogen, cortisol, aldosterone, progesterone,triiodothyronine (T₃), thyroxine (T₄), dihydroxy-vitamin D₃ (VD₃) and25-OH-cholesterol. The levels of CAT activity were plotted aspercentages of the maximal response observed in this experiment. B,Retinoic acid binding to cytosol extracts of transfected COS-1 cells.Bars represent bound ³ H-retinoic acid determined in absence (blackbars) or presence (stippled bars) of a 1000-fold excess of variouscompetitors. The values represent the mean of quadruplicatedeterminations. Competitors are retinoic acid (RA), retinol (R), T₄,dexamethasone (DEX) and vitamin D₃ (VD₃).

FIG. 3 Methods. A, CV-1 cell cotransfections and CAT assays wereperformed as described in FIG. 2. Retinoic acid was dissolved in aminimum volume of dimethyl sulfoxide and diluted in ethanol. All otherproducts were diluted in ethanol and control cultures received 0.1%solvent (v/v) in media. Dose-response curves of retinoid treatment wereperformed in triplicate. B, Subconfluent COS-1 cells were transfectedwith 10 μg/dish of a control plasmid (pRS) or pRShRR by the DEAE-Dextranmethod (see Deans, et al., (1984)). Cells were maintained for 2 days inDMEM with 5% charcoal-treated fetal calf serum, then harvested in TNE(40 mM tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA) and lysed by Douncehomogenization in hypotonic buffer (50 mM tris-HCl pH 7.4, 0.1 mM EDTA,5 mM dithiothreitol, 10 mM NaMoO₄, 10% glycerol, 0.5 mMphenylmethylsulfonyl fluoride) and centrifuged at 100,000×g for 30 minsto yield the cytosol fraction. Incubations were performed in hypotonicbuffer with 150 μg of protein from the cytosolic fraction and 2×10⁻⁸ M ³H-retinoic acid (NEN, 52.5 Ci/mmole) in a total volume of 200 μl.Specific binding was measured by the addition of 2×10⁻⁵ M ofcompetitors. Reactions were carried out at 4° C. for 16 h. Bound ³H-retinoic acid was quantitated using DE-81 filters. Reactions wereplaced on filters for 1 min and then rinsed with 5 ml of washing buffer(50 mM tris-HCl pH 7.4, 0.1 mM EDTA, 0.1 % Triton X-100). Filters weredried and counted by liquid scintillation spectrophotometry.

FIG. 4. Southern blot analysis of human genomic DNA. A, Human placentaDNA was digested with the indicated restriction enzymes. Afterseparation of the digested DNA in a 0.8% agarose gel (10 μg/lane) andtransfer to nitrocellulose filters (see Southern, (1975), the blots werehybridized with an EcoR1 X PvuII fragment from phRAR1 (.sup.˜ 600 bp)encompassing the DNA-binding domain of the hRR under high stringencyconditions (50% formamide, 5×SSPE, 1×Denhardt's, 0.1% SDS, 100 μg ml⁻¹salmon sperm DNA). The filter was washed in 0.1×SSC, 0.1% SDS at 65° C.Lambda HindIII DNA markers (size in Kb) are aligned to left of theautoradiogram. B, Analysis of human placenta DNA using the same probe asin A under non-stringent conditions. A parallel blot containingidentical samples was hybridized as in A, except that 35% formamide wasused. The filter was washed in 2×SSC, 0.1% SDS at 55° C.

FIG. 5. Northern blot analysis of retinoic acid receptor mRNA in rat andhuman tissues.

FIG. 5 Methods. Total RNA was isolated from various tissues usingguanidine thyocyanate (see Chirgwin, et al., (1980), separated on 1%agarose-formaldehyde gel, transferred to nitrocellulose, and hybridizedunder stringent conditions using the probe described in FIG. 4. Twentyμg of total RNA was used in all lanes. Migration of ribosomal RNA's (28Sand 18S) are indicated for size markers. The nitrocellulose filter wasautoradiographed at -70° C. with an intensifying screen for 1 week.

FIG. 6. Schematic amino acid comparisons of the hGR, hRR and hT₃ Rβstructures. Amino acid sequences have been aligned schematically withthe percentage amino acid identity for each region of homology in theintervals between dotted lines.

FIG. 7 is a schematic diagram of a generalized steroid thyroid retinoicacid receptor gene, showing the division of the gene into regions A/B,C, D, and E. The function of the A/B region is just beginning to beelucidated; the C region encodes the DNA-binding domain; the D region isbelieved to be a hinge region; and the E region encodes theligand-binding domain.

FIG. 8 (1 and 2) is a schematic drawing that shows amino acid comparisonof members of the steroid hormone receptor superfamily. Primary aminoacid sequences have been aligned on the basis of regions of maximumamino acid similarity, with the percentage amino acid identity indicatedfor each region in relation to the hGR (Miller et al., (1985). Domainsshown are: a domain at the NH₂ -terminal end that is required for"maximum activity"; the 66- to 68-amino acid DNA-binding domain core("DNA"); and the 250-amino acid ligand-binding (or hormone-bindingdomain) ("Hormone"). The amino acid position of each domain boundary isshown. Amino acid numbers for all receptors represent the human formswith the exception of v-erb-A and E75 (Segraves, 1988). Functionalassignments have been determined by characterization of theglucocorticoid and estrogen receptors. Designations are as follows: GR,glucocorticoid receptor; MR mineralocorticoid receptor; PR, progesteronereceptor; ER, estrogen receptor; ERR1 or ERR2, estrogen-related 1 or 2;VDR, vitamin D₃ receptor HRE hormone response element; and T₃ R.sub.βand T₃ R.sub.α, thyroid hormone receptors. The (+) or (-) indicateswhether a particular property has been demonstrated for the products ofcloned receptor cDNA or with purified receptor. This relates to whetherthe binding site has been identified structurally and whether itsenhancement properties have been demonstrated by gene transfer studies.For PR, DNA-binding properties have been shown only with the nativepurified receptor. "Hormone binding in vitro" indicates whether thisproperty has been demonstrated by translation in a rabbit reticulocytelysate system (Hollenberg et al., 1985). "Hormone binding in vivo"refers to expression of the cloned receptor in transfected cells."Chromosome" indicates the human chromosome location. Species are asfollows: h, human; r, rat; m, mouse; c, chicken; and d, Drosophilia.

FIG. 9. Structure and activity of chimeric thyroid/glucocorticoidreceptors.

FIG. 9 Methods. To construct hybrid receptors, unique NotI and XhoIsites were inserted flanking the DNA binding domains of the hGR andhTRβ. Hybrids were created by exchanging the appropriate segments of thereceptor cDNA's. "DNA" indicates the DNA binding domain; "T₃ /T₄ " and"cortisol" indicate the ligand binding domains of hTRβ and hGRrespectively. The numbers above the boxes indicate amino acid residues.Hybrids are named by letters referring to the origin of the domain; forexample, "TGT" has the amino and carboxyl termini of hTRβ and the DNAbinding domain of the hGR. All receptors were assayed on TRE-M CAT andGRE-M CAT in the absence and presence of T₃ and the syntheticglucocorticoid dexamethasone ("dex"). All of the combinations shown gaveactivation above background.

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SPECIFICATION SUMMARY

From the foregoing description, one of ordinary skill in the art canunderstand that the present invention provides substantially pure DNAwhich encodes the retinoid receptor protein referred to as retinoic acidreceptor protein. The invention also provides a plasmid containingretinoic acid receptor DNA. This plasmid, phRARl, has been depositedwith the American Type culture Collection for patent purposes.

The invention is also comprised of retinoic acid receptor proteins,including modified functional forms thereof, expressed from the DNA (ormRNA) of the invention.

In addition to novel retinoic acid receptor DNA, RNA and proteincompositions, the present invention includes chimeric hybrid receptorsmade by exchanging (1) the N-terminal domains, (2) the DNA-bindingdomains, and (3) the ligand-binding domains from hGR, hMR, hERR1, hERR2,T₃ R.sub.α, T₃ R.sub.β, RARα, and RARβ receptors with one another. Thechimeric receptors so constructed have DNA-binding domain andligand-binding domain characteristics similar to the DNA-binding domainand ligand-binding domain characteristics of the respective "parental"receptors from which they originated.

Finally, the present invention involves a bioassay for determining thefunctional ligands for receptor proteins, both wild-type and chimeric.

The phRAR1 DNA of the invention can be used to make the retinoic acidreceptor proteins, and functional modified forms thereof, in quantitiesthat were not previously possible. The same is true of the chimericreceptors. With the quantities of receptor protein available as a resultof the present invention, detailed studies can be made of both theligand/receptor complexes and the ligand/receptor/HRE complexes. Inaddition, an adequate supply of the retinoic acid receptor proteinsmeans that they can now be used to screen compounds for retinoic acidreceptor-agonists or retinoic acid receptor-antagonist activity.Availability of the receptor proteins also means that they can be usedin diagnostic assays to determine the levels of retinoic acid present invarious tissues and body fluids.

Without departing from the spirit and scope of this invention, one orordinary skill can make various changes and modifications to theinvention to adapt it to various usages and conditions. As such, thesechanges and modifications are properly, equitably, and intended to be,within the full range of equivalence of the following claims.

What is claimed is:
 1. Substantially pure DNA encoding retinoic acidreceptor wherein said retinoic acid receptor is structurally andfunctionally related to the steroid and thyroid hormone receptors. 2.Substantially pure DNA according to claim 1 wherein said retinoic acidreceptor is human retinoic acid receptor.
 3. Substantially pure DNAaccording to claim 2 wherein said human retinoic acid receptor isselected from the group consisting of human retinoic acid receptor alphaand human retinoic acid receptor beta.
 4. Substantially pure DNAencoding protein which has harmone-binding and/ortranscription-activating properties characteristic of retinoic acidreceptor wherein said retinoic acid receptor is structurally andfunctionally related to the steroid and thyroid hormone receptors. 5.Substantially pure DNA according to claim 4 wherein said protein ishuman retinoic acid receptor.
 6. Substantially pure DNA according toclaim 5 wherein said human retinoic acid receptor is selected from thegroup consisting of human retinoic acid receptor alpha and humanretinoic acid receptor beta.
 7. Substantially pure DNA sequencesselected from the group consisting of DNA sequences shown in FIGS. 1B-1,1B-2 and 1B-3.
 8. DNA encoding chimeric receptors selected from thegroup consisting of GRR, GRG, GGR, RGG, RGR, RRG, TTG, GTT, GTG, GGT,TGG, TGT, TTR, TRT, TRR, RTT, RTR, RRT, GTT, GTG, GGT, TGG, TGT, TTR,TRT, TRR, RTT, RTR, RRT, GTT, GTG, GGT, TGG, TGT, AND TTG. 9.Substantially pure DNA able to hybridize to any of the DNA's claimed inany of claims 1-8, wherein said hybridizing DNA encodes retinoic acidreceptor protein.
 10. The plasmid phRAR1 (ATCC No. 40,392).
 11. Cellstransformed by any of the substantially pure DNA's claimed in any ofclaims 10 or 1-8 or any DNA able to hybridize to said DNA's wherein saidhybridizing DNA encodes retinoic acid receptor protein.
 12. Cellstransformed by any of the substantially pure DNA's claimed in any ofclaims 10 or 1-8, or any DNA able to hybridize to said DNA's, whereinsaid hybridizing DNA encodes retinoic acid receptor protein, and whereinsaid cells contain greater than wild-type amounts of retinoic acidreceptor protein.
 13. A method for producing retinoic acid receptorprotein wherein said retinoic acid receptor protein is structurally andfunctionally related to the steroid and thyroid hormone receptors, saidmethod comprising:a. ligating substantially pure dna according to any ofclaims 10 or 1-8, or any DNA able to hybridize to said DNA's, whereinsaid hybridizing DNA encodes retinoic acid receptor protein, to areplicable expression vehicle to obtain a replicable recombinant DNAcomprising said DNA and said replicable expression vehicle; b.transforming cells of a microorganism or cell culture with saidreplicable recombinant DNA to form transformants; c. selecting saidtransformants from patent cells of said microorganism or said cellculture; d. causing said tranformants to express said DNA; e. isolatingretinoic acid receptor protein from said transformants.
 14. A replicablerecombinant DNA which comprises; a replicable vector; and substantiallypure DNA according to any of claims 10 or 1-8 or any DNA able tohybridize to said DNA's, wherein said hybridizing DNA encodes retinoicacid receptor protein.